Group B Streptococcus transcriptome when interacting with brain endothelial cells.

Bacterial meningitis is a life-threatening infection of the central nervous system (CNS) that occurs when bacteria are able to cross the blood-brain barrier (BBB) or the meningeal-cerebrospinal fluid barrier (mBCSFB). The BBB and mBCSFB comprise highly specialized brain endothelial cells (BECs) that typically restrict pathogen entry. Group B Streptococcus (GBS or Streptococcus agalactiae) is the leading cause of neonatal meningitis. Until recently, identification of GBS virulence factors has relied on genetic screening approaches. Instead, we here conducted RNA-seq analysis on GBS when interacting with induced pluripotent stem cell-derived BECs (iBECs) to pinpoint virulence-associated genes. Of the 2,068 annotated protein-coding genes of GBS, 430 transcripts displayed significant changes in expression after interacting with BECs. Notably, we found that the majority of differentially expressed GBS transcripts were downregulated (360 genes) during infection of iBECs. Interestingly, codY, encoding a pleiotropic transcriptional repressor in low-G + C Gram-positive bacteria, was identified as being highly downregulated. We conducted qPCR to confirm the codY downregulation observed via RNA-seq during the GBS-iBEC interaction and obtained codY mutants in three different GBS background parental strains. As anticipated from the RNA-seq results, the [Formula: see text]codY strains were more adherent and invasive in two in vitro BEC models. Together, this demonstrates the utility of RNA-seq during the BEC interaction to identify GBS virulence modulators. IMPORTANCE: Group B Streptococcus (GBS) meningitis remains the leading cause of neonatal meningitis. Research work has identified surface factors and two-component systems that contribute to GBS disruption of the blood-brain barrier (BBB). These discoveries often relied on genetic screening approaches. Here, we provide transcriptomic data describing how GBS changes its transcriptome when interacting with brain endothelial cells. Additionally, we have phenotypically validated these data by obtaining mutants of a select regulator that is highly down-regulated during infection and testing on our BBB model. This work provides the research field with a validated data set that can provide an insight into potential pathways that GBS requires to interact with the BBB and open the door to new discoveries.

Coxsackievirus B3 infects and disrupts human induced-pluripotent stem cell derived brain-like endothelial cells.

Coxsackievirus B3 (CVB3) is a significant human pathogen that is commonly found worldwide. CVB3 among other enteroviruses, are the leading causes of aseptic meningo-encephalitis which can be fatal especially in young children. How the virus gains access to the brain is poorly-understood, and the host-virus interactions that occur at the blood-brain barrier (BBB) is even less-characterized. The BBB is a highly specialized biological barrier consisting primarily of brain endothelial cells which possess unique barrier properties and facilitate the passage of nutrients into the brain while restricting access to toxins and pathogens including viruses. To determine the effects of CVB3 infection on the BBB, we utilized a model of human induced-pluripotent stem cell-derived brain-like endothelial cells (iBECs) to ascertain if CVB3 infection may alter barrier cell function and overall survival. In this study, we determined that these iBECs indeed are susceptible to CVB3 infection and release high titers of extracellular virus. We also determined that infected iBECs maintain high transendothelial electrical resistance (TEER) during early infection despite possessing high viral load. TEER progressively declines at later stages of infection. Interestingly, despite the high viral burden and TEER disruptions at later timepoints, infected iBEC monolayers remain intact, indicating a low degree of late-stage virally-mediated cell death, which may contribute to prolonged viral shedding. We had previously reported that CVB3 infections rely on the activation of transient receptor vanilloid potential 1 (TRPV1) and found that inhibiting TRPV1 activity with SB-366791 significantly limited CVB3 infection of HeLa cervical cancer cells. Similarly in this study, we observed that treating iBECs with SB-366791 significantly reduced CVB3 infection, which suggests that not only can this drug potentially limit viral entry into the brain, but also demonstrates that this infection model could be a valuable platform for testing antiviral treatments of neurotropic viruses. In all, our findings elucidate the unique effects of CVB3 infection on the BBB and shed light on potential mechanisms by which the virus can initiate infections in the brain.

Synthesis and Characterization of Quercetin-Iron Complex Nanoparticles for Overcoming Drug Resistance.

Quercetin, one of the major natural flavonoids, has demonstrated great pharmacological potential as an antioxidant and in overcoming drug resistance. However, its low aqueous solubility and poor stability limit its potential applications. Previous studies suggest that the formation of quercetin-metal complexes could increase quercetin stability and biological activity. In this paper, we systematically investigated the formation of quercetin-iron complex nanoparticles by varying the ligand-to-metal ratios with the goal of increasing the aqueous solubility and stability of quercetin. It was found that quercetin-iron complex nanoparticles could be reproducibly synthesized with several ligand-to-iron ratios at room temperature. The UV-Vis spectra of the nanoparticles indicated that nanoparticle formation greatly increased the stability and solubility of quercetin. Compared to free quercetin, the quercetin-iron complex nanoparticles exhibited enhanced antioxidant activities and elongated effects. Our preliminary cellular evaluation suggests that these nanoparticles had minimal cytotoxicity and could effectively block the efflux pump of cells, indicating their potential for cancer treatment.

Induced Pluripotent Stem Cell (iPSC)-Derived Endothelial Cells to Study Bacterial-Brain Endothelial Cell Interactions.

Bacterial meningitis is a serious infection of the central nervous system (CNS) that occurs when blood-borne bacteria are able to exit the cerebral vasculature and cause inflammation. The blood-brain barrier (BBB) and the meningeal blood-CSF barrier (mBCSFB) are composed of highly specialized brain endothelial cells (BECs) that possess unique phenotypes when compared to their peripheral endothelial counterparts. To cause meningitis, bacterial pathogens must be able to interact and penetrate these specialized BECs to gain access to the CNS. In vitro models have been employed to study bacterial-BEC interactions; however, many lack BEC phenotypes. Induced pluripotent stem cell (iPSC) technologies have enabled the derivation of brain endothelial-like cells that phenocopy BECs in culture. Recently, these iPSC-BECs have been employed to examine the host-pathogen interaction at the endothelial brain barriers. Using two clinically relevant human meningeal pathogens, this chapter describes the use of iPSC-BECs to study various aspects of BEC-bacterial interaction.

Group B Streptococcus-Induced Macropinocytosis Contributes to Bacterial Invasion of Brain Endothelial Cells.

Bacterial meningitis is defined as serious inflammation of the central nervous system (CNS) in which bacteria infect the blood-brain barrier (BBB), a network of highly specialized brain endothelial cells (BECs). Dysfunction of the BBB is a hallmark of bacterial meningitis. Group B Streptococcus (GBS) is one of the leading organisms that cause bacterial meningitis, especially in neonates. Macropinocytosis is an actin-dependent form of endocytosis that is also tightly regulated at the BBB. Previous studies have shown that inhibition of actin-dependent processes decreases bacterial invasion, suggesting that pathogens can utilize macropinocytotic pathways for invasion. The purpose of this project is to study the factors that lead to dysfunction of the BBB. We demonstrate that infection with GBS increases rates of endocytosis in BECs. We identified a potential pathway, PLC-PKC-Nox2, in BECs that contributes to macropinocytosis regulation. Here we demonstrate that downstream inhibition of PLC, PKC, or Nox2 significantly blocks GBS invasion of BECs. Additionally, we show that pharmacological activation of PKC can turn on macropinocytosis and increase bacterial invasion of nonpathogenic yet genetically similar Lactococcus lactis. Our results suggest that GBS activates BEC signaling pathways that increase rates of macropinocytosis and subsequently the invasion of GBS.